DESCRIPTION (appended verbatim from investigator's abstract): While the protective effects of the stress or heat shock proteins (Hsps) are well established, their role in the regulation of the apoptotic cascade is poorly understood. The proposed studies will define a framework of interactions between the stress response, induced to maintain cellular survival under adverse conditions, and apoptosis, engaged to ensure the effective deletion of damaged cells. We will investigate the role of Hsps in regulating the apoptotic machinery, using constitutive Hsc70 and its inducible counterpart, Hsp70, as follows: 1. How does Hsp70 inhibit apoptosis? Despite the well defined role of Hsp70 in mediating the cellular stress response to maintain survival, its specific impact on the apoptotic machinery is poorly understood. We have preliminary data to suggest that Hsp70 functions to prevent the assembly of a functionally competent apoptosome, by directly interacting with Apaf1 to prevent the recruitment and activation of procaspase-9. This aim will investigate the precise mechanism of Hsp70mediated inhibition of caspase activation and determine the exact nature of its interaction with the apoptosome complex. 2. How does the constitutive Hsc70 mediate the suppression of apoptosis? We have shown that Hsc70, like Hsp70, is able to inhibit caspase activation. However, dATP/ATP is able to overcome Hsc70-mediated suppression, suggesting that the regulatory mechanism differs from that of Hsp70. We propose to investigate the antiapoptotic role o Hsc70 and the co-chaperone proteins, Hsp40, Hip and Hop. This will provide an example of an integrated cellular mechanism to ensure limited basal susceptibility to damage and an inducible cellular protection in response to stress. 3. Do the antiapoptotic effects of Hsp70 impact on the function of Bcl-2 proteins? Our understanding of the apoptotic cascade suggests a likely functional cooperation between Bcl-2 and Hsp70. Bag-1, which interacts with both Hsp70 and Bcl-2 to regulate their functions, may represent a point of functional interaction between these two protein families, to regulate cellular survival. 4. What are the physiological consequences of Hsp70 mediated cellular survival? We will address the physiological impact of the antiapoptotic effects of Hsp70 in vitro, comparing cotransformation effects with those of an Apaf1 null fibroblast model and in vivo, by the development of a transgenic animal expressing Hsp70 in the lymphoid compartment. Together, our studies will secure a mechanistic basis for the function of Hsp70 in the regulation of cell death and cell survival.